Media mixing chamber

ABSTRACT

A method of reconstituting a powdered cell culture media includes providing a mixing apparatus and providing a powdered cell culture media to the fluid chamber of the mixing apparatus, prior to introducing fluid to the chamber. The method further includes introducing fluid to the chamber through an influent port, wherein the influent port is tangentially oriented relative to an inner wall of the fluid chamber to thereby cause the fluid to follow the wall of the fluid chamber in a circular motion, creating a vortex flow in the fluid chamber by introducing fluid at a sufficient flow rate, and enhancing the vortex flow with a geometric fluid flow aid by further channeling the water around the wall of the fluid chamber, around the geometric flow aid. The method further includes continuing to introduce fluid to the chamber and collecting reconstituted fluid that exits the chamber through the effluent port.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.15/087,826, filed Mar. 31, 2016, which claims the benefit of U.S.Provisional Application Ser. No. 62/257,685 filed Nov. 19, 2015, each ofwhich is incorporated herein by reference in its entirety.

BACKGROUND Field

Embodiments of the present technology generally relates to mixingapparatus for mixing at least one incoming fluid stream with a materialto be mixed with the incoming fluid stream(s). More particularly,embodiments of the present technology relate to mixing apparatusspecially adapted for reconstituting powdered cell culture media inpredetermined unit volume amounts.

Description of the Related Art

Viable animal cells and tissue in in vitro cultures have been knownsince the early 1900s. While animal cell culture today is asophisticated technology, the basic culture technique has not changedsince the beginning of the century. Cells or tissue, either primary ortransformed, are grown in a liquid nutrient mixture generally referredto as “media.” This media can be, for example, a complex mixture ofamino acids, vitamins, salts, and other components. It is oftensupplemented with 1-10% purified bovine fetal or newborn calf serum.Cell culture media and serum are available commercially from manysources.

While the basic cell culture technique has not changed appreciably overthe years, the volume of cell culture and the accessibility of thislaboratory technique continues to increase dramatically, therebychanging the needs of the cell culture technique. Not only are moreresearch laboratories, pharmaceutical and biotechnology companiesemploying tissue culture techniques but they are doing so, often, on arelatively large scale. A medical product related corporation mayconsume tens or hundreds of liters of liquid media a day and employlarge numbers of laboratory technicians and scientists to generateantibodies, growth factors or purified protein from tissue culture forcommercial use. Thus, between media supply costs and employee time thereis a considerable expense associated with the tissue culture processtoday.

Cell culture media generally is available commercially either as a drypowder which is reconstituted by adding an appropriate volume of water,or as a pre-packaged liquid. There are also a number of additives thatare typically added to the media before use. These include sodiumbicarbonate, glutamine, additional buffers or antibiotics.

The technology described herein generally relates to improved devicesand methods for mixing media in a variety of volumes and circumstances.

SUMMARY

Generally, embodiments described herein relate to devices and methodsfor powdered media that is easy to prepare, requires less storage spacethan liquid media, and/or whose preparation requires minimal effort. Thetechnology according to some embodiments relates to mixing apparatus formixing a material (e.g., a powdered media) with at least one incomingfluid stream. More particularly, some embodiments of the presenttechnology relate to mixing apparatus specially adapted forreconstituting powdered cell culture media in predetermined unit volumeamounts, as well as various methods related to the same.

The present technology represents a significant improvement in thetechnical field.

Some embodiments are based, at least in part, upon some deficienciesand/or inconveniences with existing media technologies, as recognized bythe inventors of the instant technology, or based upon the recognitionof potential improvements by the inventors.

For example, pre-packaged liquid can be sterile and aliquoted intoconvenient sizes, and may come ready to use. However, the media istypically light sensitive and has a prescribed shelf-life. Therefore,media must be ordered on a regular basis. It also should be stored underrefrigeration and, in its prepackaged form, requires significantman-power time to unpackage and transport. Further, shipping costs ofprepackaged liquid is becoming increasingly more expensive.

Furthermore, powdered media is provided in bulk or in premeasuredpackages. It tends to have a longer shelf life, is less expensive andrequires less storage space and handling time than the liquid form.However, the powdered media must be dissolved and aliquoted understerile conditions. The increased handling and preparation timeespecially for large volume media preparation often makes pre-packagedliquid media the preferred choice despite the increased cost.

Reconstitution of powdered media generally is a several step process. Toprepare a liquid media from a solid powder, a known amount of powderintended for a specific volume of media is measured out and added to avolume of distilled water which is typically slightly less than thefinal desired volume. The powder and water are stirred until the solidis completely dissolved. A specific quantity of sodium bicarbonate isadded and dissolved. The pH may thereafter be adjusted using acid orbase and additional water is added to increase the media to its finalvolume. The entire mixture is then passed through a sterilizing filter.The media may thereafter be collected in a single large sterile vessel,or proportioned into several smaller sterile vessels.

Powdered tissue culture media has a very fine particle size and ishygroscopic. When mixed with water, it has the tendency to “ball” or“clump.” Thus, when reconstituting in water or other aqueous liquid,sufficient agitation is required to break up any clumps that may formupon initial contact with water. For smaller batch sizes, sterilemagnetic stir bars can be added to the mixing container and thecontainer is then placed on a magnetic stir plate. Additionalmanipulations usually are required to add stir bars to the mixingcontainers. In a typical laboratory setting, magnetic stir plates arenot a practical solution for large volume media preparation.

In addition, due to its hygroscopic nature, the media absorbs water whenstored, especially in humid environments. Wet media has a shortenedshelf-life, becomes lumpy and requires aggressive agitation toreconstitute. Thus, powdered media shelf life could be improved if itwere provided in premeasured sealed and desiccated aliquots.

The reconstitution process requires several steps and several separatepieces of equipment. It generally requires at least one vessel, largeenough to contain the entire final volume of reconstituted media, plusone or more vessels to receive the sterile media after filtration. Thesterilized media is usually delivered into open top containers. Thus,most media preparation is done in a laminar flow hood. Processing largevolumes of media in a hood is difficult because there is often notenough space to accommodate the containers and sterile media. Someembodiments herein are based upon the recognition that a device thatwould permit the preparation of large volumes of the product withminimal physical contact and facilitate media preparation without theinconveniences described above would fulfill a long unmet need in thescientific community.

Furthermore, equipment used for reconstitution of powdered media must bethoroughly cleaned between uses to remove residue and eliminatecontaminants in the equipment. This requires significant time and evenwith careful work, the risk of contaminants remain. Thus, some of theembodiments that follow relate to a low-cost equivalent of a mediamixing chamber that is constructed for single use.

One embodiment of the technology relates to a mixing apparatus forreconstituting a powdered cell culture media. The apparatus includes atleast one fluid chamber, a first influent port at a top portion of thefluid chamber, and a second influent port at a lower portion of thefluid chamber. The apparatus further includes a geometric fluid flow aidpositioned in the fluid chamber and an effluent port at the top of thefluid chamber. A powdered cell culture media is provided in the fluid tobe mixed with a fluid provided by at least one of the first or thesecond influent port. The effluent port is configured to allowreconstituted media to exit the fluid chamber.

A second embodiment of the technology relates to a method ofreconstituting a powdered cell culture media. The method includesproviding a mixing apparatus having at least one fluid chamber, a firstinfluent port at a top portion of the fluid chamber, a second influentport at a lower portion of the fluid chamber, a geometric fluid flow aidpositioned in the fluid chamber, and an effluent port at the top of thefluid chamber. The method further includes providing a powdered cellculture media to the fluid chamber, prior to introducing fluid to thechamber, and then introducing fluid to the chamber through at least oneof the first and the second influent ports. The first and the secondinfluent ports are tangentially oriented relative to an inner wall ofthe fluid chamber to thereby cause the fluid to follow the wall of thefluid chamber in a circular motion. The method further includes creatinga vortex flow in the fluid chamber by introducing fluid at a sufficientflow rate, and enhancing the vortex flow with the geometric fluid flowaid by further channeling the water around the wall of the fluidchamber, around the geometric flow aid. The method includes continuingto introduce fluid to the chamber and collecting reconstituted fluidthat exits the chamber through the effluent port.

BRIEF DESCRIPTION OF THE DRAWINGS

The above-mentioned features, as well as other features, aspects, andadvantages of the present technology will now be described in connectionwith various embodiments of the invention, in reference to theaccompanying drawings. The illustrated embodiments, however, are merelyexamples and are not intended to limit the invention.

FIG. 1 is a schematic representation of the overall mixing chamber,sterilization filter, and receiving receptacle system in accordance withone embodiment.

FIG. 2 is a cross-sectional view of the embodiment of the mixing chamberof FIG. 1.

FIG. 3 is an exploded view of a mixing chamber according to a secondembodiment.

FIG. 4 is a cross-sectional view of a mixing chamber according to theprior art with a representation of a fluid vortex.

FIG. 5a is a top perspective view of a top cone of the mixing chamberaccording to one embodiment.

FIG. 5b is a bottom perspective view of the top cone illustrated in FIG.5 a.

FIG. 6 is a top perspective view of a bottom cone of the mixing chamberaccording to one embodiment.

FIG. 7 is a top perspective view of a lower cap of the mixing chamberaccording to one embodiment of the present invention representing theassembly of the bottom cap and the bottom cone illustrated in FIG. 6.

FIGS. 8a, 8b, and 8c are perspective views of the bottom of a middle capof the mixing chamber according to one embodiment showing the assemblyof the middle cap and the top cone illustrated in FIGS. 5a and 5 b.

FIGS. 9a, 9b, 9c, and 9d are perspective views of the bottom of a topcap of the mixing chamber according to one embodiment showing theassembly of the top cap.

FIG. 10 is a perspective view of the top cap attached to an outlet tubeaccording to one embodiment.

FIG. 11 is a perspective view of the bottom cap attached to an inlettube according to one embodiment.

FIG. 12 is a perspective view of the middle cap attached to an inlettube and a pressure transducer according to one embodiment.

FIG. 13 is a schematic representation of a single-chamber mixingapparatus in accordance with one embodiment.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part of the present disclosure. Inthe drawings, similar symbols typically identify similar components,unless context dictates otherwise. The illustrative embodimentsdescribed in the detailed description, drawings, and claims are notmeant to be limiting. The detailed description is intended as adescription of exemplary embodiments and is not intended to representthe only embodiments which may be practiced. The term “exemplary,” asused herein, means “serving as an example, instance, or illustration,”and should not necessarily be construed as preferred or advantageousover other embodiments. Other embodiments may be utilized, and otherchanges may be made, without departing from the spirit or scope of thesubject matter presented here. It will be readily understood that theaspects of the present disclosure, as generally described herein, andillustrated in the Figures, can be arranged, substituted, combined, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated and form part of this disclosure.

Embodiments described herein generally relate to devices/apparatuses,systems, and methods related to the storage, transport, preparationand/or use of media, for example, media for cell culture. One or more ofthe provided embodiments may overcome one or more of the drawbacks,limitations, or deficiencies that exist in the art, particularly withrespect to media cartridges and the limitations of such cartridges,including those with dry powder media. For example, in some embodiments,the devices/apparatuses can be single use, disposable, pre-loaded with adesired substance. The devices can include components that permit one ofmore of improved sterility, storage duration, transport, mixing ofmaterials within the devices, and use of the media. Systems and methodsof making and using the devices also are described herein.

FIG. 1 is an overall system view of one embodiment of the mixingapparatus 10, filter 58 and receiving bag 50 in accordance with thecertain embodiments. FIG. 2 is a cross-sectional view showing an innerview of the components of the mixing apparatus 10. The mixing apparatus10 comprises at least one, and in some embodiments, two chambers. Thegenerally cylindrical first chamber 12 constitutes the lower chamber inthe preferred embodiment depicted herein and a second chamber 14constitutes the upper chamber of this preferred embodiment. Fordescriptive purposes “chemical A” will refer herein to the materialcontained in first chamber 12 and “chemical B” will refer to thematerial contained in the second chamber 14 in a two chamber embodiment.

An incoming fluid stream enters the mixing apparatus 10 through at leastone influent port. It is contemplated that the features of the mixingapparatus 10 described herein are applicable in a mixing apparatus 10with one influent port, though the application will describe embodimentshaving a plurality of influent ports, such as the two influent ports 16a, 16 b shown in the embodiment of FIG. 1. The axis of the influentport(s) relative to the first chamber 12 can be such that the fluid flowenters at substantially a tangential angle to the interior wall thereof,such that liquid entering the first chamber through influent ports 16 a,16 b follows the sides of the chamber to create a circular mixing motionthat facilitates mixing of chemical A with the fluid stream within thefirst chamber. Employing more than one influent port helps direct flowtoward the undissolved volume of chemical A, typically present in asolid, powder form. Upper influent port 16 b also helps to prevent thesolid media from sticking to the top of the first chamber 12. Theinfluent ports 16 a, 16 b may be used concurrently, or can be alternatedto achieve the necessary level of fluid motion for mixing chemical Awith the fluid streams.

As chemical A dissolves in the liquid and additional liquid enters intofirst chamber 12, the liquid level advances upward through middle cap 31and enters the second chamber 14. Fluid containing chemical A passingthrough middle cap 31 and entering into the upper chamber now comes incontact with chemical B.

In a preferred embodiment, chemical B has increased solubilitycharacteristics over chemical A such that significant agitation is notnecessary to facilitate the dissolution of chemical B in liquid whichalready contains chemical A.

Liquid containing dissolved chemicals A and B thereafter exits secondchamber 14 via upper cap 41 through an effluent port 49 preferably afterpassing through a filter 48 (FIGS. 3, 9B). Liquid passing througheffluent port 49 (obscured by tubing in FIG. 1; shown in FIG. 2), in apreferred embodiment, enters into sterilization filter 58. Sterilizationfilters of the type contemplated by this technology can be purchasedfrom a number of suppliers. One example of a commercial supplier is PallCorporation, Courtland, Me. For a sterile media product, thesterilization filter apparatus will typically contain a 0.2μ filter. Thefilter may comprise nylon or cellulose acetate. Sterile liquidcontaining chemical A and chemical B thereafter exits filter 58 andpasses into a receiving receptacle 50. In some embodiments, ahydrophobic vent filter is employed between the effluent port and thesterilizing filter 58 in order to allow the air that is entrained in thedissolved medium to vent so that it does not fill the sterilizingfilter.

It is additionally contemplated that other types of filter sizes couldbe chosen for other functions. For example, the preparation ofelectrophoretic buffers requires clean, but not necessarily sterilesolutions, and a 0.45μ filter would be adequate. Similarly, thepreparation of more viscous solutions may necessitate a wider pore size.In short, the filters can be of any desired size, volume, pore size, andso forth, to accommodate the particular use of the cartridge. For otherapplications of the technology disclosed herein, no filtration apparatusneed be added. Liquid then passes directly to a receiving receptaclethrough any suitable fluid communication device, for example, flexibletubing. If a sterile filter is used, then tubing and all additionalchemicals entering multiple inlet ports 52 as well receiving receptacle50 should be sterile (see FIG. 1).

It is further contemplated that the final product may require theaddition of one or more other liquid additives, or the receptacle 50 maybe drained into a series of different containers. Therefore, one or moreinlet/outlet ports generally designated as multiple inlet/outlet ports52 are typically provided. Flow stop regulators 54 are preferablyassociated with each of the inlet ports to provide control for thesequential draining or influx of the desired additive solutions.

FIG. 3 depicts in detail an exploded view of an embodiment of mixingapparatus 10. Mixing apparatus base, or lower cap 21 is combined withfirst chamber housing 22 in association with a seal 23. A supportstructure 24 is preferably integrally molded together with or milledinto the lower cap 21 to form a ledge or lip to support the firstchamber housing 22. The support structure 24 and the first chamberhousing 22 are preferably substantially cylindrical in shape to optimizethe rotational velocity of the fluid which has been driven throughinfluent port(s) 16 under pressure. The seal 23 is preferably anelastomeric O-ring but could be a gasket or other sealing device knownto those with skill in the art. The bottom cap also supports a geometricflow aid, such as bottom cone 25. Assembly of the lower cap 21 isdescribed in more detail below with reference to FIG. 7.

First chamber housing 22 receives the inflowing liquid through one ormore influent ports 16 (shown in FIG. 1), generally tangentiallyoriented to the interior wall of the housing. The one or more influentports 16 may be integrally molded with the housing 22, or can be affixedthereto in any of a variety of ways known in the art such as byadhesive, solvent or heat bonding techniques. According to theembodiments shown, two influent ports 16 are integrally molded withlower cap 21 and middle cap 31 with a passage allowing fluid flowbetween the influent ports 16 and the interior of the housing 22. A hosebarb or other conventional connector is preferably affixed to influentport 16. Preferably, influent port 16 a is located in the lower half ofthe first chamber 12, and more preferably along the lower one-fourth ofthe first chamber 12, such as on lower cap 21. Preferably, influent port16 b is located in the upper half of the first chamber 12, and morepreferably along the upper one-fourth of the first chamber 12, such ason middle cap 31. In embodiments having a single influent port, theinfluent port may located in the lower half of the first chamber 12, andmore preferably along the lower one-fourth of the first chamber 12, suchas on lower cap 21. A protective cap may be provided to cover theinfluent port(s) thus preventing powder from spilling out prior to use.In some embodiments, quick-connect devices are employed at the influentport so that the quick-connect device prevents the egress of powderedcell culture media powder from the cartridge during storage andshipment.

Fluid entering the second port at a sufficient velocity assists thevortex created by fluid entering from the first port. For thereconstitution of large amounts of dry powder or viscous solutions, twoinfluent ports might better facilitate complete mixing. Thus, water orother solvent could be added from more than one influent port solely tosupport vortex generation. Alternatively, the liquids entering theapparatus through multiple influent ports could be of different chemicalcomposition.

The influent ports can be positioned on the same vertical plane, asshown in FIG. 1, or along different vertical planes, depending uponparticular requirements of a given application, so long as the inflowfrom port 16 a does not interfere with the inflow from port 16 b. Fluidtangentially entering the mixing chamber from both ports should flow intandem to maintain vortex activity.

It is contemplated that influent ports 16 a and 16 b have equal portdiameters. However, the diameters may be individually modified forvaried influent flow velocities. The interior diameters of each of theports and influent pressures can be varied to promote mixing of thedesired reagents. A smaller diameter port situated above a largerdiameter port would provide additional inflow velocity over the largerdiameter port. In this way an efficient vortex could be maintained tomaximize reconstitution of a given powder mixture. These design featureswill be added or included depending on the solubility of the powder in aparticular application, the volume of powder relative to the chambersize and by the chemistry required to reconstitute a given liquidpreparation.

In use, liquid enters the mixing chamber through influent ports 16 a, 16b. Faucet pressure or other inflow pressures in excess of about 1 psiare generally sufficiently strong to permit proper apparatus function.Typical tap pressure, in the area of about 25 psi is sufficient for manyembodiments. The minimum effective pressure is a function of the scaleof the first mixing chamber, the volume of chemical A contained thereinand the diameter of the influent lumen, as will be understood by one ofskill in the art. Some routine experimentation may be required tooptimize these parameters for specific applications. For example, theseparameters may be designed specific to the available water source. Whensource fluid pressure cannot be increased, the present mixing apparatusmay be designed by decreasing the diameter of the inlet.

As previously described, liquid enters the first chamber under pressureat substantially a tangent to the interior wall of the chamber. Thevelocity of the liquid entering the apparatus is determined by theincoming fluid stream pressure and can be additionally manipulated byaltering either the diameter(s) of the influent port(s) or thedimensions of the first chamber. Decreased influent port diameters willincrease the velocity of liquid entering the chamber, while increasedinfluent port diameters will decrease liquid velocity. In a preferredembodiment, a sufficient fluid velocity is achieved when the ratio ofthe cross-sectional area of the influent port through which fluid isentering the chamber (in inches) to flow rate (liters/minute) is0.0015-0.0040. In some preferred embodiments, the ratio is 0.0018 to0.0038. In some preferred embodiments, the ratio is 0.0028+/−0.0002 (inother words, 0.0028 square inches for the inlet with 1 liter per minuteof fluid flow).

It is contemplated that slight modifications of the apparatus will berequired for the proper functioning of the mixing chamber for otherapplications. For example, if the liquid is water and the product istissue culture media, then normal faucet pressure, in concert with anappropriate influent port dimension will create sufficient liquidpressure to generate the desired rotational fluid velocity. The mixingchamber influent port diameter has a direct effect on inlet velocity. Asnoted above, the inlet diameter can be increased or decreased to adjustthe velocity in order to provide adequate mixing of the media.

In an embodiment with two influent ports, such as 16 a and 16 b, it ispreferred that fluid is first provided to the chamber through upperinfluent port 16 b, so as to prevent floating of undissolved clumps ofpowdered media. Once fluid has partially filled the chamber, lower port16 a is open and begins to facilitate a vortex-like fluid flow motion.

Preferably the pressure of the liquid stream in combination with acompatible influent port diameter will provide sufficient liquidvelocity such that liquid entering the apparatus follows the surface ofthe inner chamber casing and continues along a circular pathway towardsthe center of the chamber. If the rotational fluid velocity of theliquid is sufficient, the motion subsequently establishes a turbulentvortex that serves to mix the influent liquid with the contents of thefirst chamber. This motion is shown in FIG. 4 representing the desiredfluid motion in a prior art mixing apparatus. The dashed horizontallines 80 represent the swirling fluid that creates a roughly conicalregion of air 82 at its center. The swirling vortex mixes the contentsof the chamber. Additional fluid entering the chamber pushes the vortexup the sides of the chamber and towards the top. Though this vortexmotion is anticipated under the right conditions, it is not alwaysachievable now as the powdered solid chemical has changed over time. Inmany cases, with current media, the vortex flow does not occur, and theentering fluid simply pours into the chamber. In these situations,without proper agitation, the powdered media contacts the fluid, but itdoes not fully wet to dissolve in the fluid. The clumps of powderedmedia then float, stick to the surfaces of the chamber, and/or blockinfluent or effluent ports in the chamber.

Thus, embodiments of the mixing apparatus 10 according to someembodiments help to achieve and enhance the vortex movement of the fluidby including one or more geometric flow aids, such as for example one ormore cones along the lines of bottom cone 25 and upper cone 35, withinthe volume of the first chamber 12. It should be understood that whilemultiple cones are depicted, some embodiments contemplate a single coneflow aid, while in others, additional aids can be included. Thus, asliquid flows into the chamber via the influent ports 16, it is channeledinto the chamber such that the swirling vortex motion is createdimmediately to establish the turbulent fluid motion for mixing theinfluent liquid with the contents of the first chamber 12. Furthermore,geometric flow aids further enhance mixing of the solid contents withthe influent liquid by dispersing the solid and preventing it fromsticking to or clumping at the bottom or the top of the first chamber12. The geometric flow aids preferably comprise a geometric shape thathave a decreasing diameter in a direction towards the center of thefirst chamber 12, so as to assist in the creation of the desired fluidflow vortex.

FIGS. 5A and 5B represent one embodiment of an upper geometric flow aid.In this embodiment, geometric flow aid is an upper cone 35. As describedabove, a geometric flow aid comprises a geometric shape that has adecreasing diameter in a direction towards the center of the firstchamber 12. Accordingly, upper cone 35 has conical portion 38 with adecreasing diameter towards the center of the first chamber 12. Conicalportion 38 is shown as terminating at cone tip 38 a, but it iscontemplated, in other embodiments, that conical portion my end beforereaching a tip and would have a flattened end portion. Other shapes,besides a cone, are also contemplated for the geometric flow guide.Upper cone 35 also includes a plurality of pores 36 in a pored section,through which the fluid in the first chamber 12 can pass into the upperchamber 22. The pores 36 are configured with an appropriate diameter toprevent undissolved clumps of powdered media from entering into theupper chamber 22. Finally, upper cone 35 has a base portion 37 extendingradially from a top edge of the pored section. Base portion 37 isconfigured to engage with support structure 34 of middle cap 31.

FIG. 6 represents one embodiment of a lower geometric flow aid. In thisembodiment, geometric flow aid is a bottom cone 25. As described above,a geometric flow aid comprises a geometric shape that has a decreasingdiameter in a direction towards the center of the first chamber 12.Accordingly, bottom cone 25 has conical portion 28 with a decreasingdiameter towards the center of the first chamber 12. Conical portion 28is shown as terminating at cone tip 28 a, but it is contemplated, inother embodiments, that conical portion my end before reaching a tip andwould have a flattened end portion. Other shapes, besides a cone, arealso contemplated for the geometric flow guide. Bottom cone 25 also hasa base edge 29 at the open end of the conical portion. Base edge 29 isconfigured to engage with support structure 24 of lower cap 21.

The interior of the first chamber preferably has a substantiallycylindrical configuration. This further establishes a vortex guide forthe liquid flow. Moreover, the cylinder diameter should complement theincoming fluid velocity. A first chamber diameter that is too large fora given influent flow will not support sufficient centrifugal forcealong its sides to maintain a vortex. Interior diameters that are toosmall could create excessive turbulence initially, but not form avortex, thereby potentially resulting in inadequate mixing. The diameterof the first chamber has been found to optimally be proportional to itsheight. A height to diameter ratio greater than about 2.5:1 willtypically not support the generation of a sufficiently strong vortex atinfluent flow rates of about 1-3 liters per minute. The substantiallycylindrical shape in combination with the inlet velocity and the inletangle thus combine to set up the desired vortex.

Alternatively, other chamber configurations which exhibit radialsymmetry may also be used for the first chamber housing 22. For example,spherical, hemispherical, toroidal or the like may be selected.

Some embodiments can include additional mixing or turbulence componentsthat can, for example, aid in the mixing process, or provide featuresthat assist in breaking up clumps. Such aids can include protrusions(e.g., bristles or pegs) that extend radially toward the center of thechamber, or protrusions that attach to the cones, or to a center rod andextend radially outward. In some embodiments, certain mixing aids can bespecifically excluded. For example, mixing components such aspropellers, magnets, blades, and the like, can be specifically excludedfrom some embodiments.

Referring again to FIG. 3, the upper inner surface of the first chamberhousing 22 may be coupled to, thereby enclosing the first chamber 12with, the middle cap 31. A support structure 34 is preferably integrallymolded together with or milled into the middle cap 31 to form a ledge orlip to support the first chamber 12 and the second chamber 14, as wellas other components for engaging the first chamber 12 and the secondchamber 14 and for allowing fluid flow between the chambers. Suchcomponents include the seals 32, 33, the top geometric flow aid, such asupper cone 35, and a filter disc 39. The assembly and components of themiddle cap 31 are described in more detail below with reference to FIGS.8A-8C.

The two chambers are preferably adjacent one another and separated fromone another by middle cap 31. FIGS. 1-3 illustrate a preferredembodiment where first and second chambers 12, 14 are axially aligned ina water tight seal such that liquid enters the first, or lower chamber,and moves to the second or upper chamber passing through middle cap,including circular filter disc 39. In this construction, seals 32, 33such as elastomeric O-rings are used to provide a tight seal between theupper and lower chambers. During manufacture, chemical A is preferablyplaced into first chamber 12 before the filter disc 39 has been put intoplace.

The upper chamber housing 42 is also preferably covered by upper cap 41,thereby enclosing the second chamber 14. A support structure 44 ispreferably integrally molded together with or milled into the upper cap41 to form a ledge or lip to sit upon the second chamber housing 42, aswell as for receiving other components for engaging the second chamberhousing 42 and for allowing fluid flow between the second chamber 14 andthe effluent port 49. Such components include the seals 43, 45, 46,retaining plate 47, and a filter disc 48. The assembly and components ofthe upper cap 41 are described in more detail below with reference toFIGS. 9A-9D.

FIG. 7 depicts the assembly of lower cap 21. As shown, lower capcomprises a base portion which includes influent port 16 a and a supportstructure 24 formed therein which receives bottom cone 25. Positionedabout the perimeter of bottom cone 25, and on support structure 24, isseal 23. The seal 23 is preferably an elastomeric O-ring but could be agasket or other sealing device known to those with skill in the art. Theseal 23 maintains a water-tight connection between the first chamberhousing 22 and the lower cap 21.

In the embodiment shown, lower cap 21 also serves as the base forsupport rods 64. In the embodiment of FIG. 1, it is shown that two setsof support rods 64, 65 are used to separately secure the first chamber12 and the second chamber 14, respectively. In the embodiment of FIG. 3,it is shown that a single set of rods 64 extend the full length of themixing apparatus 10. In other embodiments, it is contemplated that themixing apparatus 10 be sturdy enough to stand freely and securelywithout any support rods 64, 65.

FIGS. 8A, 8B, and 8C depict the assembly of middle cap 31. As shown inFIG. 8A, middle cap 31 comprises a base portion with a central opening.Within the central opening is support structure 34, which is configuredto receive seal 33 thereon. Seal 33 comprises a plurality of attachmentholes 33 a, each configured to receive a screw 40 therethrough to couplethe elements of middle cap 31 together. As shown in FIG. 8B, the baseportion 37 of upper cone 35 rest on the support structure 34 and theseal 33. Attachment holes 37 a are aligned with attachment holes 33 a.Finally, on top of the upper cone 35, the filter disc 39 is positioned.Filter disc 39 is secured to the middle cap 31 by positioning screw 40,or other attachment device through attachment holes 33 a and 37 a andinto the support structure 34 of the middle cap 31.

The seal 33 is preferably an elastomeric O-ring but could be a gasket orother sealing device known to those with skill in the art. The seal 33maintains a water-tight connection between the upper chamber housing 42and the middle cap 31. The filter disc 39, while preferably made ofmicroporous Porex™ plastic (Porex Technologies, Fairburn, Ga.), couldadditionally be made of porous polypropylene or polyethylene, glass,wool, micron meshing, or any of a variety of other inert substanceshaving suitable compatibility with the solvents and powders to be usedin the apparatus. Preferably, the filter material will have asufficiently small pore size to prevent escape of the powdered media. Insome embodiments, the filter has a porosity in the range of 30 to 200microns. For the preferred application described herein, the filterpreferably has a pore width of approximately 90 to 130 microns. Thefilter disc permits liquid passage into the second chamber but generallyprevents the movement of undissolved solids from the first chamber 12 tothe second chamber 14. Further undissolved solids trapped in themicroporous filter are subsequently dissolved by the continued flow offluid passing through the filter.

The first chamber housing 22 and the middle cap 31 are provided with aliquid tight seal 32 through the use of an elastomeric O-ring. The firstchamber housing 22 can either slip fit into an annular recess on the cap31 or threadably engage the base, to be coupled with the supportstructure 34. The housing can additionally be sealed to the cap 31 usingadhesives, a heat seal or other means known in the art.

FIGS. 9A, 9B, 9C, and 9D depict the assembly of upper cap 41. The viewin the figures is a bottom perspective view of upper cap 41. As shown inFIG. 9A, a seal 46 is placed to surround the exit aperture to effluentport 49. FIG. 9B depicts the placement of a filter disc 48, or effluentfilter, over the top of seal 46 and the exit aperture. FIG. 9C depictsthe placement of a second seal 45 on the inner face of filter disc 48.Finally, FIG. 9D shows placement of a retaining plate 47 covering seals45, 46 and filter disc 48, which is held in place by attachmentelements, such as screws 40, to the inner face of upper cap 41 viaengagement apertures 41 a in the upper cap. Seals 45 and 46 arepreferably used to provide a water tight seal between the upper cap 41and the upper chamber housing 42. Effluent filter 48 preferably sits atleast about one-eighth of an inch from the interior surface of upper cap41. This provides space for liquid containing chemicals A and B to passthrough the effluent filter 48 and leave via effluent port 49.

As shown in FIG. 10, effluent port comprises a hose barb connector, towhich a flexible hose is attached to move fluid from the mixingapparatus 10 to a media collector, such as receptacle 50. As shown inFIGS. 11 and 12, hoses are also preferably affixed to the influent ports16 a, 16 b and may be locked in place via a hose barb connector. In apreferred embodiment, standard flexible laboratory tubing of diametersufficiently large such that the tubing will pass over the neck of thehose barb and sufficiently small that the tubing seals over the hosebarb nozzle is employed to direct the incoming fluid stream to themixing chamber or the outgoing stream to the receptacle 50. The otherend of the influent flexible tubing is preferably applied directly to asource of fluid. In the preferred culture media application, theinfluent ports 16 a, 16 b are placed in fluid communication with adistilled deionized water (ddH₂O) source having an adapted nozzle suchas is found in most scientific laboratory ddH₂O faucets. Other tubingmaterials, nozzle adapters, and pumps may be required for use with otherwater sources or liquid solvents. Any of the ports, or other passagewaysof the mixing apparatus 10, may include a pressure transducer 17, asshown in FIG. 12. The pressure transducer can be used to quantify andmonitor the pressure in the system and make any changes that may benecessary to preserve the functionality of the system.

Flexible tubing for providing communication between system componentsmay be sterilized, such as by autoclave or gamma irradiation, andassembled together at the point of manufacture. It is additionallypreferred that a sterile receiving receptacle be supplied with theapparatus. The sterile receiving receptacle could be glass, plastic, ormetal and could be preformed or flexible. In a preferred embodiment, thereceiving receptacle comprises a sterile flexible bag such as the MediaManager Product (Irvine Scientific, Santa Ana, Calif.)

In a preferred embodiment, the mixing chambers and caps are made of anonreactive plastic polymer such as polycarbonate. Alternatively, thecaps and chambers could be molded from other plastics includingpolysulphone. Other materials include polystyrene, polyethylene,polycarbonate, plexiglass, lucite, polypropylene, a metal alloy, orglass. Preferably, the chamber housings 22, 42 will be transparent toenable visual observation of its contents or the progress of the mixingcycle.

Generally, the mixing apparatus 10 is preferably made of materials thatare appropriate for the cell culture environment, such as non-toxic,medical grade plastics or other non-toxic materials that will notcontaminate the media. In current designs, stainless steel may be used,however there is a need for single use systems. Accordingly, thematerials chosen must be relatively inexpensive, while being appropriatefor cell culture environments, to keep material and manufacture costslow for single use products.

In a preferred application, the mixing apparatus is used to preparetissue culture media. It is contemplated that the mixing chamber will beprovided prefilled with powdered media in a variety of unit volumesizes. For example, mixing chamber sizes to accommodate the preparationof 1 liter (L), 10 L, 20 L, 50 L, and as large as 100 L or larger finaltissue culture media volume are contemplated. Increasing amounts ofpowder in the lower chamber will require increased cylinder heightand/or diameter to generate a vortex of sufficient size so as tomaintain the powder in motion within the vortex until it dissolves. Inaddition, larger sizes may require a pump on the influent line togenerate sufficient influent flow to sustain a vortex. Therefore it iscontemplated that each apparatus be specifically designed to complementthe final volume of product to be prepared.

Testing has determined that a powder volume greater than about 50% ofthe chamber volume for the powdered culture media application results inpoor vortex mixing and inefficient liquid reconstitution. Testing hasadditionally determined that during operation of the mixing apparatusherein disclosed, improved reconstitution of the powder in the liquid isachieved by interrupting the inflow occasionally for approximately fiveseconds. Interrupting the flow temporarily releases pressure within thechamber thus allowing clumps of powder to draw fluid to their interior.

In addition, pressure pulsing can be applied to the mixing apparatus, toimprove reconstitution by temporarily closing the outlet valve at theeffluent port. This allows pressure to build up in the chambers whilethe valve is pinched. When the valve is released and pressure quicklydrops, the clumps of powder that may have formed in the liquid arepulverized. This pressure pulsing can be implemented manually bypinching the outlet tubing, or by incorporating a pinch valve at theeffluent port into the design.

A precalibrated receptacle 50 can be used to determine the end point ofmedia preparation. Alternatively, a predetermined volume of liquid canbe pumped through the system or a flow meter/accumulator can be used tomonitor the volume of the finished product. It is additionallycontemplated that the final volume of the liquid product can bedetermined by weight. The receiving receptacle is placed on a scale andthe receptacle is filled until the final weight of the end product isachieved.

It is important for the effective operation of the apparatus that theculture media powder remain relatively dry prior to use. Hygroscopicpowders tend to clump under humid conditions and reconstitution becomesdifficult. It is therefore contemplated that the commercial productcomprising a mixing apparatus system with powder be packaged undervacuum and/or preferably be provided with a desiccant.

It is additionally contemplated that the apparatus disclosed herein hasa number of other commercial or industrial applications. For example,many liquid pharmaceuticals are prepared in the hospital pharmacy withsome frequency and quantity. Saline solutions, alimentary preparations,imaging reagents, dyes, sterilization solutions and anesthetics arereconstituted as liquids. Premeasured aliquots provided ready forreconstitution such as contemplated by the disclosed technology canprovide an advantage over the current art.

Alternative applications include, but are not limited to, preparation ofpesticides, fertilizers, any of a variety of beverages commonly preparedfrom powder such as milk, iced tea, etc. which could all bereconstituted using the disclosed technology according to someembodiments. It is further contemplated that the liquid solventsemployed can be water, alcohols or other organics. The solubilitycharacteristics, the solvent to be used, the amount required and thechemical interactions between the solvent and the reconstitutedchemicals will serve to provide guidelines for the size of the mixingchamber and the choice of materials for the components.

A variety of modified forms of the technology can be constructed fordifferent end uses. For example, the diagrams depict a preferredembodiment wherein the first mixing chamber is coaxially aligned beneaththe second chamber and separated by a microporous circular filter disc.In this embodiment the upper and lower chambers both have a cylindricalshape and the circular filter disc follows the shape of the chambercasing. As noted, the lower chamber preferably has a generallycylindrical shape and geometric flow aids in order to facilitaterotational fluid velocity of sufficient turbulence.

However, it is not necessary for the upper chamber to have a cylindricalshape. Other shapes for the second chamber as well as for themicroporous filter disc are contemplated. The second chamber could berectangular, ovoid or essentially spherical. Further, the first andsecond chambers do not necessarily have to be positioned on top of oneanother. It is contemplated that the two chambers could be disposed sideby side or remote from one another and in fluid communication by way ofsilicone, glass or other conventional tubing.

Furthermore, it is contemplated that the mixing apparatus include onlyone chamber. A mixing apparatus 100 according to a second embodiment isshown in FIG. 13. In this embodiment, only first chamber 12 is needed.Both the powdered media and the secondary additive, such as sodiumbicarbonate, can be provided to the first chamber 12 together.Therefore, only one chamber is needed to dissolve the solid in thefluid. Accordingly, mixing apparatus 100 includes a lower cap 121,similar to lower cap 21 described above, chamber housing 122, and uppercap 131, which is similar to both middle cap 31 and upper cap 41. Inparticular, upper cap 131 includes influent port 116 b as well aseffluent port 149, and thereby serves to provide the fluid to thechamber 112 and deliver mixed fluid to a media receptacle. Other detailsassociated with mixing apparatus 10 can be applied similarly to mixingapparatus 100.

This technology according to some embodiments provides a closed,self-contained mixing system to reconstitute a unit dose of chemicalinto a known final liquid volume. The discussion provided above servesto point out those design features that can be modified to adapt thedisclosed apparatus for a wide range of applications. The desirabilityof specific influent port angles, position, number and diameter alongwith chamber dimensions, fluid pressure and a need for externalturbulence generators are design features which will be able to bereadily optimized by one of skill in the art for the reconstitution of agiven formulation.

While the preferred embodiments described herein employ powderedchemicals, it is contemplated that the mixing apparatus can work equallywell for the reconstitution of a concentrated liquid or a sequentialcombination of liquid and powder.

Thus, some embodiments of the technology disclosed herein provide amethod and apparatus for the single step preparation and, if required,sterilization of a given chemical. The system is closed, thereforehandling is minimized. All chemicals are premeasured so employeeefficiency is maximized. The closed system additionally permits acomplex sequential or multicomponent reconstitution and sterilizationprocess to be performed in a convenient location without the risk ofcontamination and with minimal variation in end product due totechnician error or batch variation. In addition, the combination of aclosed system with desiccant under vacuum yields prepackaged unitshaving a relatively long shelf life and improved tolerance totemperature change over the corresponding liquid product.

The entire content of U.S. Pat. No. 5,470,151 is herein incorporated byreference in its entirety.

The foregoing description details certain embodiments of the systems,devices, and methods disclosed herein. It will be appreciated, however,that no matter how detailed the foregoing appears in text, the devicesand methods can be practiced in many ways. As is also stated above, itshould be noted that the use of particular terminology when describingcertain features or aspects of the technology should not be taken toimply that the terminology is being re-defined herein to be restrictedto including any specific characteristics of the features or aspects ofthe technology with which that terminology is associated. The scope ofthe disclosure should therefore be construed in accordance with theappended claims and any equivalents thereof.

It will be appreciated by those skilled in the art that variousmodifications and changes may be made without departing from the scopeof the described technology. Such modifications and changes are intendedto fall within the scope of the embodiments, as defined by the appendedclaims. It will also be appreciated by those of skill in the art thatparts included in one embodiment are interchangeable with otherembodiments; one or more parts from a depicted embodiment can beincluded with other depicted embodiments in any combination. Forexample, any of the various components described herein and/or depictedin the Figures may be combined, interchanged or excluded from otherembodiments.

With respect to the use of any plural and/or singular terms herein,those having skill in the art can translate from the plural to thesingular and/or from the singular to the plural as is appropriate to thecontext and/or application. The various singular/plural permutations maybe expressly set forth herein for sake of clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims are generallyintended as “open” terms (e.g., the term “including” should beinterpreted as “including but not limited to,” the terms “comprising”and “having” should, respectively, be interpreted as “comprising atleast” and “having at least,” the term “includes” should be interpretedas “includes but is not limited to,” etc.). It will be furtherunderstood by those within the art that if a specific number of anintroduced claim recitation is intended, such an intent will beexplicitly recited in the claim, and in the absence of such recitationno such intent is present. For example, as an aid to understanding, thefollowing appended claims may contain usage of the introductory phrases“at least one” and “one or more” to introduce claim recitations.However, the use of such phrases should not be construed to imply thatthe introduction of a claim recitation by the indefinite articles “a” or“an” limits any particular claim containing such introduced claimrecitation to embodiments containing only one such recitation, even whenthe same claim includes the introductory phrases “one or more” or “atleast one” and indefinite articles such as “a” or “an.” In general, “a”and/or “an” should be interpreted to mean “at least one” or “one ormore”; the same holds true for the use of definite articles used tointroduce claim recitations. Furthermore, in those instances where aconvention analogous to “at least one of A, B, and C, etc.” is used, ingeneral, such a construction is intended in the sense one having skillin the art would understand the convention (e.g., “a system having atleast one of A, B, and C” would include but not be limited to systemsthat have A alone, B alone, C alone, A and B together, A and C together,B and C together, and/or A, B, and C together, etc.). In those instanceswhere a convention analogous to “at least one of A, B, or C, etc.” isused, in general such a construction is intended in the sense one havingskill in the art would understand the convention (e.g., “a system havingat least one of A, B, or C” would include but not be limited to systemsthat have A alone, B alone, C alone, A and B together, A and C together,B and C together, and/or A, B, and C together, etc.). It will be furtherunderstood by those within the art that virtually any disjunctive wordand/or phrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

The technology disclosed herein has numerous applications and whileparticular embodiments of the technology have been described in detail,it will be apparent to those skilled in the art that the disclosedembodiments may be modified given the design considerations discussedherein. Therefore, the foregoing description is to be consideredexemplary rather than limiting, and the true scope of the invention isthat defined in the following claims.

What is claimed is:
 1. A method of reconstituting a powdered cellculture media, the method comprising: providing a mixing apparatus,comprising: at least one fluid chamber; a first cap enclosing a lowerportion of the fluid chamber; a second cap enclosing an upper portion ofthe fluid chamber; an influent port in at least one of the first cap andthe second cap; a geometric fluid flow aid coupled to the first cap andextending away from the first cap in a direction towards a center of thefluid chamber, wherein the geometric fluid flow aid comprises ageometric shape having a decreasing diameter in the direction towardsthe center of the fluid chamber; a filter material associated with thesecond cap, wherein the filter material is used to keep the powderedcell culture media inside the fluid chamber until dissolved; and aneffluent port in the second cap; providing a powdered cell culture mediato the fluid chamber, prior to introducing fluid to the chamber;introducing fluid to the chamber through the influent port, wherein theinfluent port is tangentially oriented relative to an inner wall of thefluid chamber to thereby cause the fluid to follow the wall of the fluidchamber in a circular motion; creating a vortex flow in the fluidchamber by introducing fluid at a sufficient flow rate; enhancing thevortex flow with the geometric fluid flow aid by further channeling thefluid around the wall of the fluid chamber, around the geometric flowaid; and continuing to introduce fluid to the chamber and collectingreconstituted fluid that exits the chamber through the effluent port. 2.The method of claim 1, wherein the mixing apparatus further comprises asecond fluid chamber, the second fluid chamber being provided with apowdered additive substance, and wherein the fluid exits the firstchamber and moves into the second chamber to reconstitute the additivesubstance.
 3. The method of claim 1, wherein the mixing apparatusfurther comprises a second influent flow port in the other of the firstcap and the second cap, and wherein the method further comprisesintroducing fluid to the chamber through the second influent port. 4.The method of claim 3, wherein fluid is introduced through the influentport and the second influent port concurrently.
 5. The method of claim3, wherein the introduction of fluid alternates between the influentport and the second influent port.
 6. The method of claim 3, wherein theinfluent port is in the second cap and the second influent port is inthe first cap, and wherein the method further comprises introducingfluid through the influent port in the second cap until fluid haspartially filled the fluid chamber and then introducing fluid throughthe second influent port in the first cap.
 7. The method of claim 3,wherein the first and the second influent ports are positioned on thesame vertical plane.
 8. The method of claim 3, wherein the first and thesecond influent ports are tangentially oriented relative to an innerwall of the fluid chamber.
 9. The method of claim 1, wherein the mixingapparatus further comprises a second geometric fluid flow aid, whereinthe first geometric fluid flow aid is positioned at a bottom of thefluid chamber and the second geometric fluid flow aid is positioned atthe top of the fluid chamber.
 10. The method of claim 9, wherein thefirst and the second geometric fluid flow aids are substantiallycone-shaped.
 11. The method of claim 1, further comprising: decreasingthe diameter of an exit at the effluent port, thereby increasing thepressure inside the chamber; and returning the diameter to the normaldiameter, thereby quickly decreasing the pressure inside the chamber,and causing a pulverization of clumps of powdered media in the fluid.12. The method of claim 1, wherein the powdered cell culture media isformulated with the inclusion of sodium bicarbonate.
 13. The method ofclaim 1, further comprising determining the end point of mediapreparation by collecting reconstituted fluid in a precalibratedreceptacle.
 14. The method of claim 1, further comprising determiningthe end point of media preparation by flowing a predetermined volume ofliquid through the mixing apparatus.
 15. The method of claim 1, furthercomprising determining the end point of media preparation by monitoringthe volume of reconstituted fluid using a flow meter or accumulator. 16.The method of claim 1, wherein the powdered cell culture media isprovided pre-loaded in the mixing apparatus.
 17. The method of claim 1,further comprising disposing of the mixing apparatus after a single use.18. The method of claim 1, wherein the filter material is made fromporous polypropylene or polyethylene.
 19. The method of claim 1, furthercomprising filtering the reconstituted fluid using a sterilizationfilter.
 20. The method of claim 19, further comprising using ahydrophobic vent filter positioned between the effluent port and thesterilizing filter to allow air that is entrained in dissolved medium tovent.